Infant rhesus macaques immunized against SARS-CoV-2 are protected against heterologous virus challenge one year later

The U.S. Food and Drug Administration only gave emergency-use-authorization of the BNT162b2 and the mRNA-1273 SARS-CoV-2 vaccines for infants 6 months and older in June 2022. Yet, questions regarding the durability of vaccine efficacy, especially against emerging variants, in this age group remain. We demonstrated previously that a two-dose regimen of stabilized prefusion Washington SARS-CoV-2 S-2P spike (S) protein encoded by mRNA encapsulated in lipid nanoparticles (mRNA-LNP) or purified S-2P mixed with 3 M-052, a synthetic toll-like receptor (TLR) 7/8 agonist, in a squalene emulsion (Protein+3 M-052-SE) was safe and immunogenic in infant rhesus macaques. Here, we demonstrate that broadly neutralizing and spike-binding antibodies against variants of concern (VOC), as well as T cell responses, persisted for 12 months. At one year, corresponding to human toddler age, we challenged vaccinated rhesus macaques and age-matched non-vaccinated controls intranasally and intratracheally with a high-dose of heterologous SARS-CoV-2 B.1.617.2 (Delta). Seven of eight control rhesus macaques exhibited severe interstitial pneumonia and high virus replication in the upper and lower respiratory tract. In contrast, vaccinated rhesus macaques had faster viral clearance with mild to no pneumonia. Neutralizing and binding antibody responses to the B.1.617.2 variant at the day of challenge correlated with lung pathology and reduced virus replication. Overall, the Protein+3 M-052-SE vaccine provided superior protection to the mRNA-LNP vaccine, emphasizing opportunities for optimization of current vaccine platforms. Notably, the observed efficacy of both vaccines one year after vaccination supports the implementation of an early life SARS-CoV-2 vaccine.


Sample collection and processing
Blood was collected by peripheral venipuncture. Complete blood counts were performed on EDTA-anticoagulated blood samples, with electronic cell counts performed on a Pentra 60C+ analyzer (ABX Diagnostics) or Vet abc (SCIL Animal Care); differential cell counts were determined manually. EDTA anti-coagulated blood was used for the collection of plasma and peripheral mononuclear cells (PBMCs) as described (28). PBMCs were used fresh or stored in liquid nitrogen. Blood tubes without coagulant were also collected for processing by centrifugation (900xg for 10 minutes) for serum. Plasma and serum aliquots were stored at -70C until further processing.
For antibody analysis, saliva samples were collected with Merocel sponges (Beaver Visitec), and nasal secretions were collected with Keracel sponges (Beaver Visitec) after instilling of 250 µl PBS in the nostril and then using the sponge tip to absorb the fluid. For viral RNA analysis, nasopharyngeal, oropharyngeal and rectal secretions were collected with FLOQSwabs (Copan), placed in a vial with DNA/RNA Shield solution (Zymo Research), and stored at -70C until further processing. Nasal secretions for antibody analysis and viral RNA analysis were always collected from opposite nostrils to avoid interference. Bronchoalveolar lavages (BAL) were performed using a 20F rubber feeding tube with instillation of 20 mL sterile physiologic saline followed by aspiration with a syringe. BAL samples were centrifuged. The BAL cell pellet, together with 0.5 mL of supernatant, was then mixed with 1.5 mL of TRIzol-LS (Thermo Fisher Scientific) and cryopreserved at -70C. Additional aliquots of BAL supernatant were also immediately cryopreserved. At day 7 after challenge, animals were euthanized, and a full necropsy was performed for tissue collection, including snap-frozen tissue samples, and fixed tissues for histopathology.

Measurement of cytokines and chemokines in plasma
Plasma cytokines and chemokines were measured using the Cytokine 29-Plex Monkey Panel (Invitrogen). This is a multiplex microbead fluorescent assay utilizing the Luminex platform. The assay was run according to manufacturer's instructions.

Serum biochemistry
Biochemistry analysis on serum samples from day of challenge onwards was performed using Piccolo BioChemistry Plus disks that were run on the Piccolo Xpress Chemistry Analyzer (Abbott).
As positive controls, human monoclonal antibodies had to be used due to the lack of a rhesus macaque-specific IgG reagent of known concentration. For the D614G and B.1.617.2 assays, we used D001 (Sino Biological). Since D001 does not bind to B.1.1.529 S protein, we used M265 (Acrobiosystems). Goat Anti-human IgG HRP (Jackson Immunoresearch) was used for detection of the human mAb.

Measurement of IgG and IgA in mucosal secretions
Saliva was collected with absorbent Merocel sponges (Beaver Visitec) by placing a sponge between the cheek and gum in the back of the mouth for 5 minutes. Nasal secretions were collected with Keracel sponges (Beaver Visitec) by pipetting 100 L of 1X PBS into a nostril, briefly pressing the nostril shut and massaging it, then inserting a dry Keracel sponge. Secretions were eluted from sponges as previously described (28). A customized binding antibody multiplex assay (BAMA) was used to measure IgG and IgA antibodies to the SARS-CoV-2 Delta (B.1.617.2) receptor-binding domain (RBD) (Sino Biologicals) as described (28). Briefly, serial dilutions of standard and centrifuged secretions were mixed overnight with RBD labeled beads. Before performing IgA assays, secretions were depleted of IgG (66). The following day, beads were washed and reacted with biotinylated goat anti-human IgG (SouthernBiotech) or anti-monkey IgA (Rockland) followed by avidin-phycoerythrin (PE; Southern Biotech To assess background, a blank well of conjugated beads and a single unconjugated bead diluted in assay buffer was included on each plate. To calculate the breadth score, the mean fluorescent intensity (MFI) from blank wells was subtracted from the experimental MFI signal (background correction) and log transformed. The negative cutoff was calculated by taking the log of the mean of MFI detected in plasma samples prior to immunization (week 52) and control animals on day 0 of challenge (total of n=24) for each antigen in the assay. The magnitude of antibody binding to each antigen was calculated by taking the ratio of the background corrected, log transformed MFI of each sample and the negative cutoff; ratios >1 were assigned a score of 1. The breadth score for each animal was then calculated by multiplying the sum of the magnitudes by the average frequency for all S antigens.

Pseudovirus Antibody Neutralization Assay
SARS-CoV-2 neutralization was assessed with S-pseudotyped viruses (D614G strain and B.1.617.2) in 293T/ACE2 cells (provided by Drs. Farzan and Mu, Scripps Florida, Jupiter, FL) as a function of reductions in luciferase (Luc) reporter activity as described previously (28). Briefly, a pre-titrated dose of pseudovirus was incubated with 8 serial 5-fold dilutions of serum samples in duplicate in a total volume of 150 µL for 1 hour at 37°C in 96-well flat bottom poly-L-lysine-coated culture plates (Corning Biocoat). Cells were suspended using TrypLE Select Enzyme solution (Thermo Fisher Scientific) and immediately added to all wells (10,000 cells in 100 µL of growth medium per well). One set of 8 control wells received cells + virus (virus control) and another set of 8 wells received cells only (background control). After 66 to 72 hours of incubation, medium was removed by gentle aspiration and 30 µL of Promega 1X lysis buffer was added to all wells. After a 10-minute incubation at room temperature, 100 µL of Bright-Glo luciferase reagent was added to all wells. After 1 to 2 minutes, 110 µL of the cell lysate was transferred to a black/white plate (Perkin-Elmer). Luminescence was measured using a PerkinElmer Life Sciences, Model Victor2 Luminometer. Neutralization titers are the serum dilution at which relative luminescence units (RLU) were reduced by either 50% (ID 50 ) or 80% (ID 80 ) compared to virus control wells after subtraction of background RLUs. Serum samples were heat-inactivated for 30 minutes at 56°C prior to assay.

Whole Virus Neutralization Assay
Neutralization of SARS-CoV-2 nanoLUC carrying the D614G or B.1.617.2 mutation was assessed as described in Hou et al with modifications (67), as we reported previously (28). Briefly, under BSL-2 containment, serially diluted serum samples at 8 dilutions were incubated for one hour with SARS-CoV-2 D614G nanoLUC virus at 37°C and 5% CO 2 . After incubation, the virus/antibody mixtures were added in duplicate to black 96-well plates containing Vero E6 cells (ATCC, CRL-1586) at 2x10 4 cells per well). Each plate contains virus only (no serum) control wells. The plates were incubated for 24 hours at 37°C and 5% CO 2 . After 24 hours, cells were lysed, and luciferase activity measured with the Nano-Glo Luciferase Assay System (Promega). Neutralization activity is expressed as the dilution concentration at which the observed RLU are reduced by 50% or 80% relative to the virus-only controls.

Spike protein-expressing cell antibody binding assay (SECABA)
The cell antibody binding assay was performed based on previously described methods (68, 69), modified to use target cells derived by transfection with plasmids expressing the SARS-CoV-2 spike protein with a C-terminus flag tag. We used one plasmid expressing the D614G sequence to generate transfected 293F cells. Cells not transfected with any plasmid (mock transfected) were used as a negative control. After resuspension, washing and counting, 1x10 5 S protein paraformaldehyde. Samples were acquired within 24 hours using a BD Fortessa cytometer and a High Throughput Sampler (HTS, BD Biosciences), collecting a minimum of 50,000 total events per sample. Data analysis was performed using FlowJo v10 software (TreeStar). Gates were set to include singlet, live and flag+ events. Anti-Rhesus IgG was gated on the secondary antibody alone sample and applied to baseline/immunized RM samples to calculate %IgG+ cells. Results are reported as area under the curve (AUC) of a six dilution half-log (1:50 to 1:10,000) series calculated using the non-linear trapezoidal rule, and mock and baseline were subtracted.

Antibody-dependent natural killer (NK) cell degranulation assay
Cell-surface expression of CD107a was used as a marker for NK cell degranulation, a prerequisite process for ADCC (70, 71), performed as previously described (72). Briefly, target cells were 293T cells 2-days post transfection with a plasmid expressing SARS-CoV-2 S protein of the D614G or B.1.617.2 variants. Serum samples were tested at 1:100, 1:500, and 1:1,000 dilutions. Samples from a SARS-CoV-2 infected (PC020v1) and a non-infected (SORF Neg) human donor were tested at the same dilutions as positive and negative controls, respectively. NK cells were purified from peripheral blood of a healthy human volunteer by negative selection (Miltenyi Biotech) and were incubated with target cells at a 1:1 ratio in the presence of diluted serum, Brefeldin A (1 µL/mL, BD Biosciences), monensin (GolgiStop, 4 µL/6mL, BD Biosciences), and CD107a-FITC (0.625 µg/mL; BD Biosciences, clone H4A3) in 96-well flat bottom plates for 6 hours at 37ºC and 5% CO 2 . NK cells were then recovered and stained for viability prior to staining with antibodies specific for NK cells and cytotoxic function as previously described (72). Flow cytometry data analysis was performed using FlowJo v10 (BD Biosciences) and data are reported as the percentage (%) of CD107a + live NK cells (gating strategy: singlets, lymphocytes, aqua blue-, CD56 + or CD16 + , CD107a + ). Final data were reported as specific activity for each dilution, determined by subtraction of non-specific activity observed in assays performed with mockinfected cells and in the absence of antibodies and presented as AUC calculated using the nonlinear trapezoidal rule.

T Cell Responses
Cryopreserved peripheral blood mononuclear cell (PBMC) or lymph node cell suspensions were thawed, washed and cultured in RPMI-1640 (10 6 /mL) with 2 µg/mL overlapping peptides for the S proteins of SARS-CoV-2 wildtype (WT) / Wuhan strain, B.1.617.2, or B.1.1.529, or for SARS-CoV-2 nucleocapsid (N) protein (JPT peptides) or dimethyl sulfoxide vehicle together with costimulatory antibodies against CD28 and CD49d (BD Biosciences) (28). Positive control samples were stimulated with a cell stimulation cocktail (eBiosciences). Cells were surface stained as outlined in table S11 and then permeabilized with CytoFix/CytoPerm (BD Biosciences) per manufacturer's recommendations and stained with intracellular antibodies as recommended (table S11) and previously described (28). Data were collected using an LSRFortessa and BD FACSDiva v8.0 and analyzed with FlowJo software v10 (TreeStar). The gating strategy is illustrated in fig. S19. The percentage of cytokine positive CD4 + or CD8 + T cell responses are reported after Boolean gating and subtraction of background.

SARS-CoV-2 RNA analysis
Viral samples in RNALater were inactivated 0.1% Triton X-100 (proteomics grade, VWR) diluted in 1X phosphate-buffered saline (PBS, Life Technologies) at 37°C and with moderate shaking at 500 rpm for 30 minutes. Inactivated samples were then processed using the MagNA Pure 24 Total NA Isolation Kit (Roche Applied Science) or Direct-zol RNA Miniprep Kit (Zymo Research).
RNA was eluted using DNAse/RNase-free water. For each processing batch, positive and negative controls were used. Relative viral genome copy number was ascertained by real-time quantitative polymerase chain reaction (qPCR) using primers (N1, N3, RNP) and procedures established by the CDC (73); forward and reverse primer sequences for orf1a,b amplification were 5'-CCCTGTGGGTTTTACACTTAA-3' and 5'-ACGATTGTGCATCAGCTGA-3', respectively. The eluted RNA was subjected to reverse transcription for viral load analysis (74). cDNA was used for qPCR containing primers and SYBR green as the method of detection on a Thermo Fisher QuantStudio 7 Pro and crossing point (CP) values determined by an automated threshold method.

Lung pathology analysis
The lungs were harvested, and the trachea cannulated with an 18-gauge blunt needle. Neutral buffered 10% formalin at 30 cm fluid pressure was slowly infused into the lungs. Once fully inflated the lungs were placed into formalin and fixed for 72 hours. Then each lobe was separated and sliced from the hilus to the periphery in slabs approximately 5 mm thick. Each slice was placed in a cassette (or multiple cassettes if the slices were large), recording its place in the stack, and held in 70% ethanol until routine processing. After processing and paraffin embedding, 4 of the lobes were selected for histology (both caudal lobes, right cranial lobe and left middle lobe). Every second slab from these lobes was sectioned at 5 µm for hematoxylin and eosin (H&E) staining.
Depending on the size of the lung lobe, between 20 and 24 slides per animal were blindly and independently evaluated by two pathologists. Each slide was given a single score from 0 to 4 which encompassed the extent and severity of the interstitial and alveolar inflammation, such as "interstitial pneumonia", as described in tables S7 and S8.   (see table S1) of the Protein (blue symbols) group, mRNA group (orange symbols), or control RMs (gray symbols); horizontal lines indicate the group median value. Differences within a group between two timepoints were determined by Wilcoxon rank test and differences between RMs of different groups at the same timepoint by Mann-Whitney test, with *p<0.05 and **p<0.01.   (see table S1) and horizontal lines indicate the group median value. Differences between two groups were determined by Mann-Whitney test with *p<0.05, **p<0.01, and ***p<0.001. expression is listed in parentheses above the x-axis for each of the testing days. The dashed horizontal lines indicate the lower limit of detection. Blue, orange, and gray symbols represent RMs of the Protein, mRNA, or control group, respectively. Differences in RNA concentrations between two groups were assessed by Mann-Whitney test with *p<0.05 and **p<0.01.      on the day of challenge (day 0) and on day 7 after challenge. Note that plasma antibodies in the single RM of the control group with a breadth score >8 on day 0 did not bind to SARS-CoV-2 nucleoprotein (see Fig. 3; binding to S proteins of variants of concern was confirmed in a repeat sample. (B) Median antibody binding was measured as log 10 median fluorescence intensity (MFI) to the B.1.617.2 S protein on day 0 and day 7 after challenge. Individual RMs of the two vaccine groups are presented by their respective symbol listed in table S1. Differences between the two timepoints were determined by Wilcoxon rank test with *p<0.05. Data for n=8 RMs per group and timepoint are shown in each of the graphs. RMs are presented by their respective symbol listed in table S1. Differences between the two timepoints were determined by Wilcoxon rank test with *p<0.05. Boolean gating. Horizontal bars indicate medians, each symbol represents an individual RM as outlined in table S1. Differences between RMs of the same group at different timepoints were determined by Wilcoxon rank test with *p<0.05 and **p<0.01. Differences in T cell responses between two groups were determined by Mann-Whitney test with *p<0.05.

Fig. S14. Correlation between lung inflammation and virus replication. Lung virus replication
(log 10 copies/30 mg) was positively correlated with lung pathology scores as determined by Spearman rank test. The limit of detection of the viral qRT-PCR assay was 5 copies/30 mg tissue (dashed horizontal line). Each symbol represents an individual RM as outlined in table S1; n=8 RMs per group.   IgG specific activity, and peripheral blood CD4 + and CD8 + T cell responses. Challenge outcome parameters included the lung pathology score, lung viral RNA, nasal and pharyngeal viral RNA reported as AUC over the 7-day post-challenge period, the sedated and cage-side clinical observation scores, and the day 2 increase in systemic plasma cytokines. Data for each parameter across the 3 groups were tabulated and then the 25 th , 50 th , and 75 th percentiles were calculated. The actual data for each RM were then color-coded based on their percentile as shown in the legend. In addition, data were scored based on their percentile with data below the 25 th percentile being assigned a score of "1", between the 25 th and 50 th percentile a score of "2", between the 50 th and 75 th percentile a score of "3", and a score of "4" for data >75 Th percentile.
The data were entered into a matrix and ordered from lowest (no or mild) to highest (severe) challenge outcome score. A separate matrix was assembled for WT/D614G-specific immune     a MIP-1, macrophage inflammatory cytokine-1 alpha; RANTES, Regulated upon Activation, Normal T Cell Expressed and Secreted; I-TAC, interferon-inducible T cell alpha chemoattractant; MDC, macrophage-derived chemokine; MIF, macrophage migration inhibitory factor; G-CSF, granulocyte colony stimulating factor; GM-CSF, granulocyte macrophage CSF; VEGF, vascular endothelial growth factor; EGF, epidermal growth factor; HGF, hepatocyte growth factor; FGF-2, fibroblast growth no interstitial pneumonia 0% -alveolar parenchyma within normal limits 1 minimal interstitial pneumonia <5% -rare small regions of mild alveolar interstitial expansion (1 to 2 cells thick) 2 mild interstitial pneumonia 5% to 25% -some regions of increased interstitial cellularity by inflammatory cells (up to 2 or 3 cells thick) -usually with slightly increased numbers of alveolar macrophages 3 moderate interstitial pneumonia 25% to 50% -moderate expansion of alveolar septae by inflammatory cells (with areas up to 4 to 6 cells thick) -increased numbers of alveolar macrophages and sometimes alveolar neutrophils -some cases of mild type 2 hyperplasia 4 severe interstitial pneumonia >50% -marked increase in expansion of alveolar septae by inflammatory cells (with some areas more than 6 cells thick) -increased numbers of alveolar macrophages and usually some alveolar neutrophils -often type 2 hyperplasia -some cases of alveolar edema or rare alveolar fibrin accumulation